经典HLA基因遗传变异对汉族人群严重脓毒症易感性影响的关联研究和机制探讨

The dysfunction of innate and adaptive immune responses plays an important role in the pathophysiology of severe sepsis. Antigen presentation is a pivotal process in the body's immune system by which macrophages, dendritic cells and other cell types capture antigens and then enable their recognition by T-cells. HLA genes encode molecules that are central to the host immune response and antigen presentation, and variation in peptide-binding grooves of the classical HLA genes likely affects severity and outcome after infection with pathogen. There is a growing consensus that specific HLA alleles and heterozygosity at HLA class I and II loci can exert a strong impact on progression and outcome in inflammatory, autoimmune, and infectious disease. However, until now there was no report to evaluate the association between HLA gene variation and severe sepsis. Recently, we found that HLA-DR gene might be one of the susceptibility genes to severe sepsis by PCR-SBT（Sequencing based typing）methods. Moreover, the most interesting finding was that the HLA-DR gene variation was associated with the HLA-DR expression levels in severe sepsis patients. In the future study, we will perform deep sequencing in the classical HLA I and HLA II genes using Roche-454 GS Flx Platform to explore the association between classical HLA gene variation and susceptibility, severity and outcome of severe sepsis. Finally, we will conduct functional experiments to analysis the influence of the associated variation on immune response in the severe sepsis patients. Our study will guide our understanding of how HLA risk alleles influence the immune repertoire and disease susceptibility; hence give help to develop immune regulation in disease therapy.

固有免疫和适应性的免疫系统调节失控引起的免疫功能紊乱是严重脓毒症发病的主要病理生理机制。HLA 分子参与的抗原呈递是适应性免疫反应的关键环节，其肽结合槽区氨基酸组成和空间结构的差异，决定了不同个体对同一抗原应答能力的差异。近年来相继发现经典HLA I和II类基因变异与个体免疫反应的程度相关，与多种感染性和自身免疫性疾病的易感性相关。HLA基因遗传变异是否与严重脓毒症的发生和发展相关，至今尚未见报告。近期我们利用PCR-SBT技术，对HLA-DR基因进行分型，发现HLA-DR基因变异与严重脓毒症易感性和患者早期的免疫功能状态显著相关。本课题拟利用高通量、先进的二代测序技术，通过对经典HLA I和II类基因多态性进行分型，系统的探讨其基因遗传变异与严重脓毒症易感性、病情程度和预后的相关性；并借助体内外功能研究，探讨HLA 遗传变异对严重脓毒症早期免疫功能状态的影响，为个体化的免疫调理提供参考。

固有免疫和适应性的免疫系统调节失控引起的免疫功能紊乱是严重脓毒症发病的主要病理生理机制。HLA 分子参与的抗原呈递是适应性免疫反应的关键环节，其肽结合槽区氨基酸组成和空间结构的差异，决定了不同个体对同一抗原应答能力的差异。近年来相继发现经典HLA I和II类基因变异与个体免疫反应的程度相关，与多种感染性和自身免疫性疾病的易感性相关。HLA基因遗传变异是否与严重脓毒症的发生和发展相关，至今尚未见报告。围绕上述问题，主要开展了以下内容的研究：①单核细胞HLA-DR表达和炎症/抗炎症细胞因子浓度的变化与脓毒症预后的关联研究：在国内外首次构建了脓毒症免疫状态的分型；并且开展了单中心、前瞻性的观察研究，发现HLA-DR表达与脓毒症患者的病情程度和预后有关，与脓毒症患者的二次感染有关；②HLA-I和II类区域基因变异与脓毒症病情程度和预后的关联研究：首次开展了脓毒症的GWAS研究，重点关注了HLA区域基因变异与脓毒症易感性和预后的关联研究，发现了HLA-DRB1*1501基因变异与HLA-DR的表达有关，与患者的预后有关；并在18染色体区域发现一个新的基因变异与脓毒症患者的易感性相关；③HLA-DRB1* 1501与HLA-DR表达的相关性研究：根据是否携带HLA-DRB1*1501将患者分为HLA-DRB1*1501阳性组与阴性组。结果发现阳性组的HLA-DR水平明显低于阴性组，而死亡率却远远高过阴性组（70% vs 5.68%，p<0.001）。同时，阳性组的疾病严重程度、部分炎症标志物与细胞因子的水平都要高过阴性组。存活组患者的HLA-DR水平一直高于同时间点的死亡组患者，但无论存活组还是死亡组，HLA-DR在各时间点上的动态变化并不显著；④另外利用候选基因的研究策略进行了凝血通路关键基因SNPs与脓毒症的易感性研究，研究发现编码蛋白C和TF的基因变异与脓毒症致ARDS的易感性有关，并通过功能研究证实了其发挥作用的机制。以上研究结果进一步阐释了脓毒症患者的免疫状态存在动态变化，而且与患者的预后相关；HLA基因变异与脓毒症的发病有关，影响脓毒症的预后，为将来脓毒症的临床分型提供了依据。